Fig. 4

Evaluating bias in sample composition. a Metapolyzyme lyses FFPE bacteria. (i) Bar plot showing quantitative PCR DNA recovery after lysis (cyan)/no lysis (grey) with Metapolyzyme. Increase in recovery is shown above each test. For each bar, n = 6. Treatment with 100 μg of Metapolyzyme for 4 h markedly increased the recovery of DNA in all tests (p < 0.001) as per Wilcoxon signed-rank test. (ii) Immunofluorescence microscopy images of Protoblocks stained with DAPI (blue) for 4T1 cells, α-E. coli (green) and α-S. aureus (red). Protoblocks were fixed for 24 h. b Measuring bias introduced by host DNA. (i) Box plot comparing DNA recovery of bacteria in Protoblocks loaded with (cyan) and without 4T1 cells (orange). Quantitative PCR recovery was normalised to a sample input of 10⁶ cells. For each box, n = 6. Protoblocks without 4T1 cells had a higher recovery of all bacteria taxa. Difference of means between tests was measured using a Wilcoxon signed-rank test, for all bacterial taxa. (ii) Immunofluorescence microscopy images of Protoblocks with and without mammalian cells, stained with α-E. coli (green) and DAPI (blue) for 4T1 cells. Protoblocks were fixed for 48 h. c Testing host DNA depletion strategies. DNA recovery of 4T1 cells (orange), Escherichia (cyan) and Staphylococcus (green) after a 10-min treatment with either Triton-X (0.1%), Saponin (0.1%) or Molysis CM buffer. For each bar, n = 3. % increase or decrease in recovery from untreated is shown above each bar. Dotted lines indicate the PCR recovery of samples without host depletion. (In all cases, p = + < 0.1, * < 0.05, ** < 0.01 and *** < 0.001)